The Genetics of

Heidi L. Rehm, Ph.D. Harvard Medical School Boston, MA Faculty Disclosure Information

In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation

This presentation will not include discussion of pharmaceuticals or devices that have not been approved by the FDA or "off-label" uses of pharmaceuticals or devices. Causes of Childhood Hearing Loss

Environmental or Unknown Etiology

Syndromic Genetic Nonsyndromic

Autosomal Recessive Mitochondrial Autosomal X-Linked Dominant Human Karyotype We inherit two copies of every gene, one from each parent. http://www.accessexcellence.org/AB/GG/ Dominant Mutations

• Dominant hearing loss can be caused by only one copy of a mutated gene

• Dominant hearing loss is seen in every generation

• If a parent has a dominant mutation, each child has a 50 % chance of inheriting it. Autosomal Recessive Mutations

• For recessive hearing loss, both copies of a gene must be mutated to get hearing loss.

• Often, there is no family history of hearing loss.

• Each child will have a 25% chance of hearing loss.

A carrier is a person who carries one copy of a recessive mutation , but does not have hearing loss. X-Linked Recessive Mutations

• Only males are affected.

• Each son will have a 50% chance of having hearing loss.

• Each daughter has a 50% chance of being a carrier. Mitochondrial Mutations

• Only the mother passes mitochondria to her children.

• All children will inherit a mitochondrial mutation from their mother.

• Mitochondrial mutations are often variable in their expression of hearing loss.

DFNA6 DFNA2 DFNA14 DFNB9 DFNA38 DFNA37 DFNB25 DFNA13 DFNB6 DFNA5 DFNB32 DFNA43 AR DFNA7 DFNA18 DFNA27 DFNA22 DFNB4 DFNA37 DFNB27 DFNB15 DFNA24 DFNA1 DFNB14 DFNB36 DFNB26 DFNA15 DFNA21 DFNA28 DFNA34 DFNA16 DFNA44 AR DFNB17 DFNA39 DFNA10 DFNB13 DFNA42

DFNA32 DFNB30 40/92DFNB18 NonsyndromicDFNA31 DFNB11 DFNB1 DFNA9 DFNB23 DFNA48 DFNB7 DFNB2 DFNA3 DFNB5 DFNA40 DFNA19 DFNA11 DFNA25 DFNB16 DFNA47 Deafness Genes IdentifiedDFNA23 DFNB22 DFNA51 DFNB12 DFNA8/12 DFNA41 DFNA30 DFNA36 DFNB21 DFNB35 DFNB31 DFNB24 DFNB33 DFNB20 DFN6 DFN4 DFNB19 DFNB3 DFNB15 DFN3 DFN2 DFNA20 DFNA4 DFNB8/10 DFNA26 DFNB28 Syndromic Hearing Loss

Syndromes Gene(s) Alport COL4A5, COL4A3, COL4A4 Branchio-Oto-Renal EYA1 Jervell and Lange-Nielsen KCNQ1, KCNE1/IsK Mitochondrial (MELAS/MERRF) tRNAleu(UUR),tRNAlys Neurofibromatosis type II NF2 Norrie NDP Osteogenesis Imperfecta COL1A1, COL1A2 Pendred PDS Stickler COL2A1, COL11A2, COL11A1 Tranebjaerg-Mohr (DFN1) DDP Treacher Collins TCOF1 Usher MYO7A, USH1C, CDH23, PCDH15, SANS, USH2A, VLGR1, USH3 Waardenburg PAX3, MITF, SLUG, EDNRB, EDN3, SOX10 There are currently over 400 syndromes with associated hearing loss. UsherUsher SyndromeSyndrome (3-6% of childhood deafness) Hearing Vestibular Retinitis Loss System Pigmentosa Type I Congenital Congenital Onset pre- profound balance puberty problems Type II Congenital Normal Onset in mild-severe teens-20s sloping Type III Progressive Progressive Variable later onset balance onset problems Usher Type Locus Gene Relative Incidence USH1A 14q32 unknown 2%

USH1B 11q13.5 MYO7A 60% USH1C 11p15.1 USH1C 5% USH1D 10q CDH23 10% USH1E 21q unknown Rare

USH1F 10q21.1 PCDH15 Rare

USH1G 17q24-25 SANS Rare

USH2A 1q41 USH2A (+51) 80% USH2B 3p23-24.2 unknown Rare

USH2C 5q14.3-q21.3 VLGR1 15%

USH3 3q21-q25 USH3 100% Jervell & Lange-Nielsen Syndrome

• Incidence: 1/250,000 • Phenotype – Severe-profound congenital sensorineural hearing loss – Prolonged QT interval – Syncope – Arrythmia – Sudden death • Heart condition diagnosed by EKG and treatable with beta-blockers DFNB4DFNB4 HearingHearing Loss/Loss/PendredPendred SyndromeSyndrome

• Congenital sensorineural hearing loss w/ EVA or Mondini • ~20% with late onset goiter -> 10% hypothyroid • Incidence: ~5% of congenital hearing loss

• Inheritance: Autosomal Recessive? Photograph of mild goiter provided by Richard JH Smith, MD.

Pendred Syndrome Normal HearingHearing LossLoss andand EVAEVA

~30% of sporadic cases and ~90% of pts with family hx have mutations in the PDS gene.

Patients with 2 mutations will likely develop thyroid disease whereas those with only 1 or 0 mutations will likely not (Pryor et al. J Med Genet. 2005. 42(2):159- 65) Universal Newborn Hearing Screening

Audiology – Diagnostic ABR

Otolaryngology – Clinical Work-Up

Physical Exam Ophthalmology

Medical Hx (pre, peri, postnatal) Clinical Genetics Family History EKG Labs: Serology, culture, urinalysis CT/MRI Gene Tests Cx26, Others

Management and Intervention Genetic Counseling Cochlear Implant Hearing Aids ASL Causes of Childhood Hearing Loss

Environmental or Unknown Etiology

Syndromic Genetic Nonsyndromic

Autosomal Recessive Mitochondrial Autosomal X-Linked Dominant Cx26 Should all Children with Hearing Loss have Cx26 Testing?

? Even cases that have an apparent explanation for hearing loss still may have Cx26 mutations

? Case A: Congenital

? Case B: CMV perinatal infection

? Case C: Prematurity

? Case D: Hyperbilirubinemia GJB2 - Connexin 26 Exon 1 ~ 3000 bp Exon 2 (681 bp) DFNB1 (Recessive) Mutations Missense: M1V, T8M, G12V, K15T, S19T, I20T, R32C, M34T, V37I, A40E, A40G, G45E, E47K, W77R, V84L, L90P, V95M, H100Y, S113R, delE120, K122I, R127H, R143W, E147K, P175T, R184P, R184W, S199F, L214P, 05INS4, I203K, N206S, S139N, H100, E101G, L90V, M93I, 486INST, Q80R, I82M, S85P, A88S, L174R, L79P, Q80P, S19T, I20T, V27I+E114G, R32L, R165W Nonsense: W24X, W44X, E47X, Q57X, Y65X, Y97X, Q124X, Y136X, W112X, W172X, C64X, Q80X, E147X Frameshift: 31del14, 31del38, 35delG, 35insG, 51del12insA, 167delT, 176del16, 235delC, 269insT, 299delAT, 314del14, 333delAA, 290linsA, 310del14, 312del14, 509del14, 509insA, 515 del17, 631delGT, 504insAAGG, 515del17, 572delt, 645delTAGA, 302del3, 469delG Splice Site: IVS1+1G>A GJB6-D13S1830 (Cx30) Deletion

DFNA3 (Dominant) Mutations delE42, W44S/C, R75Q, D179N, R184Q, C202F, M163L w/ Skin Disease G12R, S17F, D50N, N54K, G59A, D66H, R75W, R75Q Unknown Significance H73R, R98Q, R127C, K224Q, IVS1, N54I, V84A, S85Y, 313delAA, 314delA, 360delG, T123N, E129K, R143Q, Y155X, M163V, A171T, F191L, A197S Polymorphisms V27I, F83L, E114G, T123A, V153I, G160S, C169Y, I203T, Exon1 - 493del100, -3558T, -1C>T, I30I, A40A, R127H, S72C, Q80Q, L89L, R104R, I128I, V182V, V190V, 682C>T, 765C>T Connexin 26 Gene Sequence

ATGGATTGGGGCACGCTGCAGACGATCCTGGGGGGTGTGAACA AACACTCCACCAGCATTGGAAAGATCTGGCTCACCGTCCTCTTC ATTTTTCGCATTATGATCCTCGTTGTGGCTGCAAAGGAGGTGTG GGGAGATGAGCAGGCCGACTTTGTCTGCAACACCCTGCAGCCA GGCTGCAAGAACGTGTGCTACGATCACTACTTCCCCATCTCCCA CATCCGGCTATGGGCCCTGCAGCTGATCTTCGTGTCCAGCCCA GCGCTCCTAGTGGCCATGCACGTGGCCTACCGGAGACATGAGA AGAAGAGGAAGTTCATCAAGGGGGAGATAAAGAGTGAATTTAAG GACATCGAGGAGATCAAAACCCAGAAGGTCCGCATCGAAGGCT CCCTGTGGTGGACCTACACAAGCAGCATCTTCTTCCGGGTCATC TTCGAAGCCGCCTTCATGTACGTCTTCTATGTCATGTACGACGG CTTCTCCATGCAGCGGCTGGTGAAGTGCAACGCCTGGCCTTGT CCCAACACTGTGGACTGCTTTGTGTCCCGGCCCACGGAGAAGA CTGTCTTCACAGTGTTCATGATTGCAGTGTCTGGAATTTGCATC CTGCTGAATGTCACTGAATTGTGTTATTTGCTAATTAGATATTGT TCTGGGAAGTCAAAAAAGCCAGTTTAA Hearing Loss Severity Associated with Biallelic Cx26 Mutations 45 40 M34T or V37I 35 30 25 20 15

Number of Ears 10 5 0 Normal Mild Moderate Severe Profound

Data from Children’s Hospital Boston (Kenna and Rehm, 2005) Pure Tone Averages for M34T and V37I Genotypes

M34T/ M34T/ M34T/35delG M34T V37I V37I/V37I

0 X X Normal 20 X X X X X X X X X X X X X X Mild 40 X X X

60 X Moderate X 80 Severe

100 Profound 120 Cx26 Gene Test Outcomes

1. Two copies (homozygous) of a single mutation e.g. 35delG/35delG 2. Two different mutations (compound heterozygous) e.g. 35delG/167delT 3. No Cx26 mutations detected 4. Only one mutation detected (heterozygous) e.g. 35delG/+ Cx26 Gene Testing at Children’s Hospital Boston

Testing results from deaf probands:

Biallelic mutations: 20/101 (20%) Heterozygous mutations: 12/101 (10%) No mutation detected: 71/101 (70%)

Kenna and Rehm et al, 2000 Explanations for Deafness in an Individual with a Single Cx26 Mutation

? The mutation may act dominantly (There are at least six Cx26 mutations known to act dominantly.)

? The Cx26 mutation is unrelated to the deafness (The deafness may be caused by another gene mutation or a non-genetic cause.)

? The test did not detect the second mutation (There may be a mutation in a non-coding region.)

? The genetic background of the patient may alter the mutations ability to cause deafness 2 Cases of Delayed Onset Cx26 Deafness

? Two children who passed hearing screens later developed hearing loss

? Each was found to be have 2 mutations in the Cx26 gene (both were 35delG/35delG)

? Child 1: Diagnosed with profound deafness at age 15 months - (normal newborn ABR)

? Child 2: Diagnosed with severe hearing loss at age 9 months - (normal at 5 months) JAMA 284 (10):1245, 2000. Progression of Cx26 Hearing Loss

Progressive 19%

Stable Profound 43% 38%

Total = 100 patients

Hair Cell Stimulation

Sound wave Hair Cell Transduction

K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+

K+ K+ K+ K+ Potassium Recycling in the

Endolymph K+ K+ K+ K+ K+ K+ K+

K+

Is Cx26 hearing loss always nonsyndromic??

Most likely, but that does not guarantee the absense of unrelated medical problems Major Causes of Hearing Loss

Anatomical Traumas/ Infections Exposures Drugs Unknown

Syndromic Genetic~50 Genes Nonsyndromic

Autosomal Recessive Mitochondrial Autosomal X-Linked Dominant Cx26 ~50 Genes tRNAser tRNAser(UCN) 12S rRNA GJB2 A1555G The longer the gene CLDN14 Cx26 the more difficult GJB6 and expensive to GJB3 Cx30 POU4F3 develop and perform POU3F4 POU3F4 TMPRSS3 a genetic test. DFNA5 COCH COCH EYA4 SLC26A4 (PDS) KCNQ4

Genes PDS DIAPH1 OTOF COL11A2 MYH9 TECTA MYO7A CDH23 MYO15 0 2000 4000 6000 8000 10000 12000 Coding Sequence Length (bp) Benefits of Genetic Testing for Deafness

Genetic testing can:

? Aid in diagnosis and determining prognosis

? Eliminate the need for further clinical testing

? Help predict (or rule out) the onset of other clinical features of a syndrome (e.g. blindness in )

? Help make more informed treatment decisions

? Aid in making reproductive choices

? Supply considerable “psychological” contentment Genes and the Environment

Mitochondrial 12S rRNA gene A1555G, C1494T, 961delCins(T)n mutations = increased risk of hearing loss from aminoglycoside antibiotics (i.e. gentimicin, tobramycin, amikacin)

Benefit of test: Prevent other family members with the mutation from being exposed to aminoglycosides Note: Hearing loss due to these mutations can occur without aminoglycosides and aminoglycosides can cause hearing loss without these mutations. Drawbacks of Genetic Testing for Deafness

Genetic testing may:

? Not always give clear answers or any answer

? Put a psychological burden on a parent or relative

? Put an individual at risk for discrimination

? Create ethical dilemmas associated with reproductive choices Attitudes Towards Genetic Testing

Middleton et al. 1998, Attitudes of Deaf Adults toward Genetic Testing for Hereditary Deafness 55% of “Deaf Nation” attendees thought genetic testing would do more harm than good, 46% thought its use devalued deaf people Brunger et al. 2000, Parental Attitudes toward Genetic Testing for Pediatric Deafness 96% of parents of deaf children had positive attitude toward genetic testing Middleton et al. 2001, Prenatal Diagnosis for Inherited Deafness 21% (deaf), 39% (HOH), 49% (hearing) would consider prenatal testing – 6%, 11% and 16% would consider terminating pregnancy Genetic Counseling Study

Brunger et al. 2000 found that all respondents had a poor understanding of genetics.

We are examining the extent to which families are receiving genetic counseling for hearing loss and how well they understand the genetics of hearing loss. Did you have post-test genetic counseling and who provided it?

Counseling

Counseling with a Genetic Professional 100 90 80 70 60 50 40 Percentage 30 20 10 0 Pilot Study Cx26 Study Relationship between Genetic Counseling with a Genetics Professional and Average Quiz Score

6

5 Genetic Counseling with GP

No Genetic Counseling with GP 4

3

2 Average Quiz score

1

0 Pilot Study Cx26 Study Quiz Question #6

There are many genes that cause hearing loss and tests are not yet available for all of these genes. Therefore, even if a person’s current genetic test results are negative, the hearing loss may still be genetic. Were you aware of this fact?

? Yes ? No

Pilot study - 34% said “No” Cx26 Study – 22% said “No” Take Home Message

Genetic counseling is equally if not more important for those families who are NEGATIVE for Cx26 and other tests ? Could still be genetic ? Recurrence risk 10-15% ? Follow for future testing Version 2

Gene Test Cards: Facts About Genetic Testing Connexin 26 Test Mitochondrial Tests SLC26A4 (PDS) Test COCH Test Connexin 30 Deletion Test

http://hearing.harvard.edu CommonCommon CausesCauses ofof HearingHearing LossLoss FOR PARENTS & FAMILIES

Department of Otolaryngology ? Children’s Hospital Boston

HARVARD MEDICAL SCHOOL Acknowledgements

Margaret A. Kenna, MD

Anna Frangulov

Contact information: http://hearing.harvard.edu For booklets: [email protected] For me: [email protected] Right Ear

Frequency (Hz) 250 500 1000 2000 4000 8000 -10 0 Right Ear 7.5 year old girl 10 discovered with 20 10/28/1999 30 12/13/1999 moderate hearing loss 40 1/14/2000 during school exam 4/3/2000 50 5/2/2000 60 5/11/2000 70 No newborn hearing 5/24/2000 Hearing Level (dB) 80 5/30/2000 screen was performed 90 6/8/2000 100 110 Left Ear 5/27/00 – Patient 120 Frequency (Hz) reported sudden loss 250 500 1000 2000 4000 8000 -10 of hearing 0 Left Ear 10 Serial 6 20

30 days apart shows 25- 40 30 dB 50 60

70 Cx26: 35delG/101del2 Hearing Level (dB) 80

90

100

110

120